CN102068833A - Polyamine modified antenna type polymer capillary electrochromatography open tubular column and preparation method thereof - Google Patents
Polyamine modified antenna type polymer capillary electrochromatography open tubular column and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a polyamine modified antenna type polymer capillary electrochromatography open tubular column and a preparation method thereof. The open tubular column is prepared by the steps of capillary inner wall vinylation, epoxy group grafting reaction in the column, branched imine open loop grafting reaction, and the like. The capillary open tubular column stationary phase is a metacrylic acid ester antenna type polymer. Polyamine is bonded on the surface of the metacrylic acid ester antenna type polymer in the capillary open tubular column, and the bonded polyamine is branched polyethyleneimine. The bonded phases of the polyamine antenna type polymer modified capillary open tubular column are uniformly distributed; a plurality of amino groups are bonded and modified on the tube wall; and the invention provides obvious and stable anode electroosmotic flow and more electrostatic interaction sites, effectively reduces the adsorbing effect on macromolecular samples, such as protein, and the like, has high repeatability and is suitable for capillary electrochromatography microseparation of polypeptides and proteins in complex biological samples.
Description
Technical field:
The present invention relates to whisker type polymer capillary vessel electric chromatogram open tubular column of polyamine species modification and preparation method thereof, belong to the capillary electric chromatogram technical field.
Background technology:
Capillary electric chromatogram (capillary electrochromatography) be developed recently get up a kind of novel chromatogram differential is from technology efficiently, fast.Capillary separation column is the core of CEC, the capillary open tubular column is as one of main type of capillary column, compare with packed column and need not to prepare stopper, compare the mixed proportion that need not accurate optimization polymer and pore-foaming agent with integral post, have that preparation is simple, operation easily, equilibration time is short, flushing is convenient and the little characteristics of sample size, can obtain higher post and imitate.
Because effectively the post surface area of bonded functional group is less in the open tubular column, comparing of open pipe electrochromatography is low, and column capacity is less.In order to overcome a low difficult problem of comparing, scientist modifies etching tube wall bonding, porous layer in succession, the polymerization of high aglucon capacity is applied to fixing preparation mutually with technology such as nanometer-material-modified, to improve the open tubular column capacity.The whisker type polymer has widely in liquid chromatogram and membrane technology field because possessing higher aglucon capacity to be used, and post is compared, part capacity and specific area all are significantly improved, and separating effect obviously improves.
Branched polyethylene imine is the polymer that has primary amine, secondary amine and tertiary amine group and part side chain, and easy and epoxy, acid, isocyanate compound react, and can carry out the number of chemical modification.Branched polyethylene imine positive ion density and active high, Heat stability is good, and have polar group (amino) and hydrophobic group (alkyl, vinyl) structure, and can combine with different materials, be a kind of good capillary function decorative material.That the present invention proposes first is vinylated via capillary tube inner wall, graft reaction in the epoxide group post, at capillary tube inner wall bonding epoxide group, the grafting branched polyethylene imine prepares fixedly phase of open tubular column on this basis, make prepared open tubular column whisker type polymerization fixedly significantly promote in phase useful effect site, obvious and stable anode EOF and more electrostatic interaction site is provided, can effectively reduce big suction-operateds that divides subsample such as protein, significantly improve open tubular column capacity and separating effect.Simultaneously, under EOF pattern in the same way, acid compound also can be realized separation rapidly and efficiently, and separate object scope applicatory is wider.
Summary of the invention:
In order to realize that open tubular column fixedly significantly promotes in phase useful effect site, stable anode EOF is provided, the invention provides whisker type polymer capillary vessel electric chromatogram open tubular column of polyamine species modification and preparation method thereof.Adopt steps such as capillary tube inner wall is vinylated, the interior graft reaction of epoxide group post, branched polyethylene imine ring-opening reaction, promptly at first introduce the vinyl of reactable at inner surface of tube wall by methyl allyl acyloxypropyl trimethoxysilane, implement graft reaction in the epoxide group post then, form the surface by vinyl and GMA polymerisation and have epoxy radicals, be the methacrylate polymers that whisker type distributes; To use branched polyethylene imine and epoxy reaction at last, with the branched polyethylene imine molecular linkage in capillary surface, thereby form stable polyethylene imine beautify layer.Prepared polymerization is a large amount of amine groups of phase bonding fixedly, and the whisker type structure makes the useful effect site significantly promote, thereby can provide obvious and stable anode EOF and more electrostatic interaction site, stability is high, can effectively reduce the adsorption effect of alkaline matter at tube wall, be applied to biological samples such as protein, peptide and separate, have good effect.Simultaneously, under EOF pattern in the same way, acid compound also can be realized separation rapidly and efficiently, and separate object scope applicatory is wider.
The objective of the invention is to be achieved through the following technical solutions:
The whisker type polymer capillary vessel electric chromatogram open tubular column that one polyamine species is modified, described capillary open tubular column bonding has methacrylate whisker type polymer, and the polymer surfaces bonding has polyamines, and the polyamines of institute's bonding is a branched polyethylene imine.
The preparation method of the whisker type polymer capillary vessel electric chromatogram open tubular column that described polyamines is modified may further comprise the steps:
1, capillary tube inner wall is vinylated:
With volume content is 30～50% the methyl allyl acyloxypropyl trimethoxysilane and the N of corresponding ratio, after dinethylformamide and 1.0mg/mL polymerization inhibitor diphenylpicrylhydrazine mix, sonic oscillation 15～20min injects through HCl solution and the pretreated capillary of NaOH solution at low temperatures.With the capillary closed at both ends, under 120 ℃, add thermal response 6～12h or 40～60 ℃ of following reaction 12～14h, be preferably 120 ℃ of reaction 12h down.After question response is finished, use N successively, dinethylformamide and methyl alcohol wash capillary column 30min respectively, dry up in 90 ℃ of nitrogen.
2, graft reaction in the epoxide group post
With volume content is 10% GMA and 90% dry toluene and 0.25～1.25mg/mL initator azodiisobutyronitrile, concentration is preferably 0.75mg/mL, after the mixing, sonic oscillation 15～20min at low temperatures, nitrogen blowing 5～10min removes the oxygen that dissolves in the mixture, then mixture is injected the capillary of vinylated processing.With the capillary closed at both ends, be dipped in 80 ℃ of water-baths and react 2～20h, after question response is finished, wash capillary column 30min respectively with toluene and methyl alcohol successively, dry up in 90 ℃ of nitrogen.
3, branched polyethylene imine open loop bonding
With volume content is that 5～35% branched polyethylene imine is dissolved in the phosphate buffer, the pH of phosphate buffer is 6.0, concentration is 20～50mmol/L, at room temperature behind sonic oscillation 15～20min, mixture is injected the capillary of bonding epoxide group behind the mixing.With the capillary closed at both ends, be dipped in and react 12～24h or room temperature reaction 24～48h in 80 ℃ of water-baths, after question response is finished, successively with deionized water, methyl alcohol and the mobile capillary column that washes respectively mutually, remove the residual reagent in the post, obtain the whisker type open pipe electric chromatographic column of polyethylene imine beautify.
Advantage of the present invention:
1, the fixing steps such as capillary tube inner wall is vinylated, the interior graft reaction of epoxide group post, branched polyethylene imine ring-opening reaction that adopt mutually of open tubular column of the present invention, the whisker type polymer fixedly on the basis of phase again the grafting branched polymer be prepared from, unique whisker type fixedly phase structure has been realized a large amount of amino extensive distributions in the tube wall space, useful effect site, space increases, specific area increases, and helps improving open tubular column capacity and separating effect.
2, open tubular column of the present invention is fixing adopts the method multistep reaction of chemical bonding to form mutually, owing to be that chemical bond combines with the capillary wall surface silanol group, the stability and the reappearance of post are better, and bonded phase distributes more even, be not prone to obscission, column life is longer.
3, polyamines of the present invention modify the whisker type polymer fixedly phase surface have polar group (amino) and hydrophobic group (chain-like alkyl) structure, separation selectivity improves, stronger and stable anode EOF and more action site can be provided in wider pH range, can be applicable to the open pipe capillary electric chromatogram, be suitable for polypeptide class and protein-based separation in the complex biological sample.Also can satisfy acid compound separation requirement rapidly and efficiently under the EOF pattern in the same way, it is wider to be suitable for the separate object scope.
Description of drawings:
The whisker type polymer capillary vessel electric chromatogram open tubular column that Fig. 1, polyamines are modified is the phase structure formula fixedly.
The whisker type polymer capillary vessel electric chromatogram open tubular column that Fig. 2, polyamines are modified separates enkephalins
1：Tyr-Gly-Gly-Phe-Met，2：Tyr-Gly-Gly-Phe-Leu，3：Gly-Gly-Phe-Met，4：Gly-Gly-Phe-Leu，5：Tyr-Gly-Gly，6：Gly-Phe-Met，7：Gly-Phe-Leu
The whisker type polymer capillary vessel electric chromatogram open tubular column isolated protein that Fig. 3, polyamines are modified
1: myoglobins, 2: ALA, 3: bovine serum albumin(BSA), 4: lysozyme.
The whisker type polymer capillary vessel electric chromatogram open tubular column separating nucleotide that Fig. 4, polyamines are modified
1:5 '-uridylic acid, 2:5 '-guanylic acid, 3:5 '-adenylate, 4:5 '-cytidine monophosphate.
The specific embodiment:
Embodiment 1: the preparation of the whisker type polymer capillary vessel electric chromatogram open tubular column that polyamines is modified
1. capillary tube inner wall is vinylated
The adding volume content is 30% methyl allyl acyloxypropyl trimethoxysilane and 70% N in advance with the pretreated capillary of the NaOH solution of the HCl solution of 0.1mol/L and 1.0mol/L, the mixture of dinethylformamide and 1.0mg/mL diphenylpicrylhydrazine reacts 12h down in 120 ℃.After question response is finished, use N successively, dinethylformamide and methyl alcohol wash capillary column 30min respectively, dry up in 90 ℃ of nitrogen.
2, graft reaction in the epoxide group post
With volume content is after the dry toluene of 10% GMA and 90% and 0.75mg/mL azodiisobutyronitrile mix, sonic oscillation 15min at low temperatures, nitrogen blowing 5min removes the oxygen that dissolves in the mixture, then mixture is injected the capillary of vinylated processing.With the capillary closed at both ends, be dipped in 80 ℃ of water-baths and react 14h, after question response is finished, wash capillary column 30min respectively with toluene and methyl alcohol successively, dry up in 90 ℃ of nitrogen.
The open loop of 3 branched polyethylene imines is modified
With volume content be that 30% branched polyethylene imine is dissolved in that pH is 6.0, in the 50mmol/L phosphate buffer, sonic oscillation 15min at room temperature behind the mixing injects the capillary of bonding epoxide group with mixture.With the capillary closed at both ends, be dipped in 80 ℃ of water-baths and react 12h, after question response is finished,, remove the residual reagent in the post successively with deionized water, methyl alcohol with flow and wash capillary column mutually respectively, obtain the whisker type open pipe electric chromatographic column of polyethylene imine beautify.This open tubular column fixedly surface bond of phase has the amino and hydrophobic chain-like alkyl of a large amount of polarity, and fixedly phase structure is as shown in Figure 1 for it.
Embodiment 2: the whisker type polymer capillary vessel electric chromatogram open tubular column that polyamines is modified separates enkephalins
Under the capillary electric chromatogram pattern, with pH 2.5, ion concentration 30mmol/L, acetonitrile: phosphate (25/75, v/v) buffer solution is the phase that flows, electrokinetic injection voltage 18kV, time 4s, add separation voltage 18kV, under the condition of wavelength 214nm, 7 kinds of similar enkephalins class materials of isoelectric point are carried out electrochromatography separation-ultraviolet detect on the polymer-modified open tubular column of branched polyethylene imine whisker type, as shown in Figure 2, eluting peak is followed successively by: 1 Tyr-Gly-Gly-Phe-Met, 2Tyr-Gly-Gly-Phe-Leu, 3 Gly-Gly-Phe-Met, 4 Gly-Gly-Phe-Leu, 5 Tyr-Gly-Gly, 6 Gly-Phe-Met, 7Gly-Phe-Leu, realize baseline separation, eliminated the conditions of streaking that the suction-operated of peptide on tube wall causes.
Embodiment 3: the whisker type polymer capillary vessel electric chromatogram open tubular column isolated protein that polyamines is modified
Under the capillary electric chromatogram pattern, with pH 2.5, ion concentration 20mmol/L, acetonitrile: phosphate (20/80, v/v) buffer solution is the phase that flows, manual potential difference sample introduction height 10cm, time 5s, add separation voltage 18kV, under the condition of wavelength 214nm, four kinds of protein are effectively separated on the whisker type polymer open tubular column of this polyethylene imine beautify, as shown in Figure 2, eluting peak is followed successively by: 1 myoglobins, 2 ALAs, 3 bovine serum albumin(BSA)s, 4 lysozymes, except the bovine serum albumin(BSA) peak width is big, other protein peak shapes are symmetry, does not produce hangover, has effectively avoided the adsorption phenomena of protein-based large biological molecule at tube wall.
Embodiment 4: the whisker type polymer capillary vessel electric chromatogram open tubular column separating nucleotide that polyamines is modified
Under the capillary electric chromatogram pattern, with pH 3.0, ion concentration 20mmol/L, acetonitrile: phosphate (25/75, v/v) buffer solution is the phase that flows, electrokinetic injection voltage 18kV, time 5s adds under the condition of separation voltage 18kV, wavelength 254nm, and the monophosphic acid nucleotide class polar compound of four kinds of acidity reaches efficient separation in the 8min under the EOF pattern in the same way on the whisker type polymer open tubular column of this polyethylene imine beautify.Its spectrogram as shown in Figure 4.
Claims (2)
1. the whisker type polymer capillary vessel electric chromatogram open tubular column modified of a polyamine species, it is characterized in that: described capillary open tubular column is fixing to be methacrylate whisker type polymer mutually.Methacrylate whisker type polymer surfaces bonding in the described capillary open tubular column has polyamines, and the polyamines of institute's bonding is a branched polyethylene imine.
2. the preparation method of the whisker type polymer capillary vessel electric chromatogram open tubular column modified of polyamines according to claim 1 is characterized in that comprising following steps:
(1) capillary tube inner wall is vinylated:
With volume content is 30～50% the methyl allyl acyloxypropyl trimethoxysilane and the N of corresponding ratio, after dinethylformamide and 1.0mg/mL polymerization inhibitor diphenylpicrylhydrazine mix, sonic oscillation 15～20min injects through HCl solution and the pretreated capillary of NaOH solution at low temperatures.With the capillary closed at both ends, under 120 ℃, add thermal response 6～12h or 40～60 ℃ of following reaction 12～14h, be preferably 120 ℃ of reaction 12h down.After question response is finished, use N successively, dinethylformamide and methyl alcohol wash capillary column 30min respectively, dry up in 90 ℃ of nitrogen.
(2) graft reaction in the epoxide group post
With volume content is 10% GMA and 90% dry toluene and 0.25～1.25mg/mL initator azodiisobutyronitrile, concentration is preferably 0.75mg/mL, after the mixing, sonic oscillation 15～20min at low temperatures, nitrogen blowing 5～10min removes the oxygen that dissolves in the mixture, then mixture is injected the capillary of vinylated processing.With the capillary closed at both ends, be dipped in 80 ℃ of water-baths and react 2～20h, after question response is finished, wash capillary column 30min respectively with toluene and methyl alcohol successively, dry up in 90 ℃ of nitrogen.
(3) branched polyethylene imine open loop bonding
With volume content is that 5～35% branched polyethylene imine is dissolved in the phosphate buffer, the pH of phosphate buffer is 6.0, concentration is 20～50mmol/L, at room temperature behind sonic oscillation 15～20min, mixture is injected the capillary of bonding epoxide group behind the mixing.With the capillary closed at both ends, in 80 ℃, react 12～24h or room temperature reaction 24～48h, after question response is finished, successively with deionized water, methyl alcohol and the mobile capillary column that washes respectively mutually, remove the residual reagent in the post, obtain the whisker type open pipe electric chromatographic column of polyethylene imine beautify.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104436758A (en) * | 2014-12-09 | 2015-03-25 | 兰州大学 | Aminated composite coating as well as preparation method and application thereof |
| CN108686634A (en) * | 2011-10-19 | 2018-10-23 | 生物辐射实验室股份有限公司 | The solid phase of hybrid chromatogram purification for protein |
| CN110302562A (en) * | 2019-07-17 | 2019-10-08 | 武汉轻工大学 | A kind of preparation method of molecular engram open tubular column |
-
2010
- 2010-10-27 CN CN 201010524084 patent/CN102068833A/en active Pending
Non-Patent Citations (3)
| Title |
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| 《 Electrophoresis》 20091231 Liang Xu et al. Novel negatively charged tentacle-type polymer coating for on-line preconcentration of proteins in CE 689-695 2 , * |
| 《Anal. Chem》 19970701 Emilio Co´ rdova et al. Noncovalent Polycationic Coatings for Capillaries in Capillary Electrophoresis of Proteins 1370-1379 1-2 , * |
| 《Electrophoresis》 20080229 Liang Xu et al. Novel open tubular CEC with tentacle-type polymer stationary phase functionalized by phenylalanine 880-888 1-2 , * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108686634A (en) * | 2011-10-19 | 2018-10-23 | 生物辐射实验室股份有限公司 | The solid phase of hybrid chromatogram purification for protein |
| CN108686634B (en) * | 2011-10-19 | 2021-03-16 | 生物辐射实验室股份有限公司 | Solid phase for mixed mode chromatographic purification of proteins |
| CN104436758A (en) * | 2014-12-09 | 2015-03-25 | 兰州大学 | Aminated composite coating as well as preparation method and application thereof |
| CN104436758B (en) * | 2014-12-09 | 2016-04-06 | 兰州大学 | A kind of amination composite coating and its preparation method and application |
| CN110302562A (en) * | 2019-07-17 | 2019-10-08 | 武汉轻工大学 | A kind of preparation method of molecular engram open tubular column |
| CN110302562B (en) * | 2019-07-17 | 2021-08-27 | 武汉轻工大学 | Preparation method of molecular imprinting open tubular column |
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Application publication date: 20110525 |